1. Field of the Invention
The invention relates to growth media for vascular lineage cells, particularly media using oligosaccharide-based hydrogels as a substrate for vascular lineage cell growth and morphogenesis.
2. Background of the Art
Generating a functional vascular network has the potential to improve treatment for vascular disease and successful organ transplantation. In recent decades, postnatal vasculogenesis has been considered to be an important mechanism for neovascularization via circulating endothelial progenitor cells (EPCs) derived from marrow (Asahara et al., Science, vol. 275, pp. 964-967, 1997; Hill et al., New England J. Med., vol. 348, pp. 593-600, 2003). Since their discovery, marrow derived circulating endothelial progenitor cells (EPCs) have been considered to participate in postnatal vasculogenesis (Asahara et al., 1997; Hill et al., 2003; Urbich et al., Circ. Res., vol. 95, pp. 343-353, 2004). Putative EPCs have been proposed as a potential therapeutic tool for treating vascular disease, either through infusion to the site of vascularization (Schatteman et al., J. Clin. Invest., vol. 106, pp. 571-578, 2000; Silva et al., Proceedings of the National Academy of Sciences, vol. 105, no. 38, pp. 14347-14352, 2008) or ex vivo expansion for engineering vascularized tissue constructs (Shepherd et al., FASEB J., vol. 20, pp. 1739-1741, 2006; Au et al., Blood, vol. 111, pp. 1301-1305, 2008; Melero-Martin et al., Circulation Research, vol. 103, no. 2, pp. 194-202, 2008). It has become more evident that endothelial colony-forming cells (ECFCs), a subtype of EPCs recently indentified from circulating adult and human umbilical cord blood, expressed characteristics of putative EPCs (Yoder et al., Blood, vol. 109, pp. 1801-1809, 2007; Hirschi et al., Arterioscler. Thromb. Vasc. Biol., vol. 28, pp. 1584-1595, 2008; Yoder, M C, Journal of Thrombosis and Haemostasis, vol. 7, SUPPL. 1, pp. 49-52, 2009). These ECFCs are characterized by robust proliferative potential in forming secondary and tertiary colony as well as de novo blood vessel formation in vivo (Yoder, M C, Arterioscler. Thromb. Vasc. Biol., vol. 30, no. 6, pp. 1094-1103, 2010; Ingram et al., Blood, vol. 105, no. 7, pp. 2783-2786, 2005).
Differentiation, mobilization, and recruitment of EPCs and endothelial cells (ECs) are regulated foremost by vascular endothelial growth factor (VEGF) (Asahara Circ. Res., vol. 85, pp. 221-228, 1999; Li et al., FASEB J., vol. 20, pp. 1495, 1497, 2006). Administration of VEGF into the site of ischemia has been reported to induce ECs mobilization and restore blood flow (Kava et al., J. Cereb. Blood. Flow. Metab., vol. 25, pp. 1111-1118, 2005; Sun et al., J. Clin. Invest., vol. 111, pp. 1843-1851, 2003). The extracellular matrix (ECM) provides critical support for ECs; their adhesion to the ECM is required for their proliferation, migration, morphogenesis, and survival—as well as, ultimately, for the stabilization of blood vessels (Davis et al., Circ. Res., vol. 97, pp. 1093-1107, 2005),—through both biochemical and mechanical functions (Deroanne et al., Cardiovasc. Res., vol. 49, pp. 647-658, 2001; Sieminski et al., Cell Biochem. Biophys., vol. 49, pp. 73-83, 2007); Mammoto et al., Nature, vol. 457, pp. 1103-1108 2009). Matrix elasticity has been reported to induce stem cell differentiation and morphological changes (McBeath et al., Dev. Cell., vol. 6, pp. 483-495, 2004; Engler et al., Cell, vol. 126, pp. 677-689, 2006). Changes in physical interactions between cell surface integrins and the ECM, due to alterations in ECM elasticity, regulate cell shape and cytoskeletal structure (Ingber et al., J. Cell Biol., vol. 109, pp. 317-330, 1989; Matthews et al., J. Cell Sci., vol. 119, pp. 508-518, 2006; Chen et al., Science vol. 276, pp. 1425-1428, 1997). Mechanical forces exerted by ECs on the matrix stimulate capillary growth in vivo (Moore et al., Dev. Dyn., vol. 232, pp. 268-281, 2005) and formation of capillary-like structures (CLSs) in vitro (Davis et al., Exp. Cell Res., vol. 216, pp. 113-123, 1995; Ingber et al., Cell, vol. 58, pp. 803-805, 1989). Recently, matrix elasticity has been reported to modulate the expression of VEGF receptor 2 (VEGFR2) (Mammoto et al., Nature, vol. 457, pp. 1103-1108, 2009), and biomechanical forces alone were sufficient to mediate vascular growth in vivo independent of endothelial sprouting (Kilarski et al., Nat. Med., vol. 15, pp. 657-664, 2009).
Tube morphogenesis is essential for the development of a functional circulatory system [24,25]. Longitudinal vacuoles that appeared to be extruded and connected from one cell to the next were first described by Folkman and Haudenschild (Folkman et al., Nature, vol. 288, pp. 551-556, 1980). These observations were confirmed and extended by later studies showing that intracellular vacuoles arise from events downstream of integrin-ECM signaling interactions, where lumen formation is mediated through the activation of the Rho GTPase Cdc42 (Kamei et al., Nature, vol. 442, pp. 453-456, 2006; Bayless et al., J. Cell Sci., vol. 115, pp. 1123-1136, 2002; Davis et al., Exp. Cell Res., vol. 224, pp. 39-51, 1996; Iruela-Arispe et al., Dev. Cell, vol. 16, pp. 222-231, 2009). Moreover, at the site of neovascularization, activated membrane type 1-matrix metalloproteinase (MT1-MMP) activates pro-MMPs at the pericellular area, which digest the ECM and allow EC migration and tubulogenesis (Collen et al., Blood, vol. 101, pp. 1810-1817, 2003; Gálvez et al., J. Biol. Chem., vol. 276, pp. 37491-37500). Such matrix remodeling by MMPs is implicated in various pathological conditions including atherosclerosis, inflammation, and ischemia (Romanic et al., Stroke, vol. 29, pp. 1020-1030, 1998; Galis et al., J. Clin. Invest., vol. 94, pp. 2493-2503, 1994).
Vascular regeneration and repair are complex processes, which require EPCs to break down the extracellular matrix (ECM), migrate, differentiate, and undergo tube morphogenesis. In the last decades understanding of the role of ECM in vascular morphogenesis has been widely expanded due to well defined in vitro angiogenesis models. Natural ECM such as matrigel, collagen, and fibrin gels have been widely used to study the molecular mechanisms that regulate endothelial cells (ECs) tubulogenesis (Davis et al., Birth Defects Research Part C—Embryo Today: Reviews, vol. 81, no. 4, pp. 270-285 2007; Kniazeva et al., Am. J. Physiol. Cell Physiol., vol. 297, no. 1, pp. C179-187, 2009), as well as to transplant vascular progenitor cells, such as ECFCs (Critser et al., “Collagen matrix physical properties modulate endothelial colony forming cell-derived vessels in vivo.” Microvascular Research, In Press) and EPCs and mesenchymal stem cells (Au et al., Blood, vol. 111, pp. 1302-1305, 2008; Au et al., Blood, vol. 111, no. 9, pp. 4551-4558, 2008; Melero-Martin et al., Circulation Research, vol. 103, no. 2, pp. 194-202, 2008) to generate vascular networks in vivo. However, the inherent chemical and physical properties of these natural materials have limited their manipulation for vascular tissue engineering.
The ECM contains instructive physical and chemical cues required for a delicate balance between various factors and cells to guide vascular assembly (Davis, G E, Am J Physiol Heart Circ Physiol, vol. 299, pp. H245-H247, 2010; Sacharidou et al., Blood, vol. 115, no. 25, pp. 5259-5269, 2010; Deroanne et al., Cardiovasc. Res., vol. 49, pp. 647-658, 2001; Sieminski et al., Cell. Biochem. Biophys., vol. 49, pp. 73-83, 2007; Kniazeva et al., Am. J. Physiol. Cell Physiol., vol. 297, no. 1, pp. C179-187, 2009; Mammoto et al., Nature, vol. 457, pp. 1103-1108, 2009; Stratman et al., Blood, vol. 114, no. 2, pp. 237-47, 2009). Depending on their spatial and temporal distribution throughout the body, each ECM component can have a different role in angiogenesis. HA and fibronectin, which are major components of embryonic ECM, are vital vascular regulators during embryogenesis (Toole, B P, Semin. Cell. Dev. Biol., vol. 12, no. 2, pp. 79-87, 2001; Toole, B P, Nat Rev Cancer, vol. 4, no. 7, pp. 528-539, 2004); while, collagen and laminin, which are abundant in adult ECM, are crucial for maintaining vascular homeostasis in adults (Davis et al., Current Opinion in Hematology, vol. 15, no. 3, pp. 197-203, 2008). Natural ECM, like collagen, fibrin, and matrigel, has been used both as model to study vascular morphogenesis and as scaffold to deliver vascular construct. However the use of natural ECM hydrogels has been limited due to their inherent physical and chemical properties. Moreover, their clinical usage has been hampered due to problems associated with complex purification processes, pathogen transfer, and immunogenicity.